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Notes: Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells.
A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR. (3289)

Notes: The authors of this article describe using the SV 96 Total RNA Isolation System on the Beckman-Coulter BioMek® FX automated laboratory instrument to purify total RNA from 96 separate 2 x 105 HUVEC cell cultures. To demonstrate the quality and consistency of RNA yield, the isolated RNA was reverse transcribed and used in real-time PCR. (3090)

Notes: The SV 96 Total RNA Isolation System was use to purify total RNA from stabily transfected MCF-7 cell clones expressing deletion mutants of SEL1L, a protein implicated in breast tumor formation. One microgram of total RNA purified with the SV 96 Total RNA Isolation System was then used in RT-PCR. (2846)

Notes: The SV 96 Total RNA Isolation System was used to isolate total RNA from human umbilical vein endothelial (HUVEC) cells, primary keratinocytes (NHEK-adults) and human rheumatoid arthritis synovial fibroblasts (RASF) infected with adenovirus expressing siRNAs targeted at a variety of messages. The isolated RNA was used in real-time SYBR Green RT-PCR reactions to investigate the amount of message present after infection. For reverse transcriptase reactions, the researchers used 5-100ng of purified total RNA. Results were normalized to GAPDH message levels. (2752)

Notes: The SV Total RNA Isolation System was used to isolated RNA from various dissected mouse brain areas. Ten microliters of the isolated RNA were used for RT-PCR analysis of gene expression patterns. (2186)

Notes: The SV Total RNA Isolation System was used to isolated RNA from the syncytiotrophoblast of human term placentas. The isolated RNA was used for RT-PCR with M-MLV Reverse Transcriptase used for the RT reaction. The 110 kDa PKD2 protein was expressed in vitro with the TNT® T7 Coupled Reticulocyte Lysate System with and without Canine Pancreatic Microsomal Membranes. The channel activity of the expressed protein was examined and the functional channel could be inhibited by known inhibitors. Expressing the luciferase control with the membranes did not produce the same effect. (2187)

Notes: The SV Total RNA Isolation System was used to isolate RNA from transiently transfected COS-7 cells bearing expression plasmid for exon 6 from a variety of patients. The isolated RNA was used for RT-PCR analysis. (2185)

Notes: Jurkat cells were fractionated into polysomes, and the resulting polysomes were ethanol precipitated. Total RNA was isolated from the polysomes with the SV Total RNA Isolation System. The system was also used to isolate RNA directly from Jurkat cells without fractionation. The isolated RNA was used for Northern blots, dot blots, RT-PCR and RNase protection assays. (2182)

Brain Res. Mol. Brain Res.76, 25–35.
Expression of the GDNF family members and their receptors in the mature rat cochlea.2000

Stöver, T., Gong, T.L., Cho, Y., Altschuler, R.A. and Lomax, M.I.

Notes: Total RNA was isolated from various rat tissues with the SV Total RNA Isolation System. Yields are reported as 10µg from 16 whole cochlea, 8µg from 16 modiola, 10.4µg from 48 cochlear sensorineural epithelial/lateral walls and 50µg from the substantia nigra region of four brains. The isolated RNA was used for RT-PCR in the presence of RNasin® Ribonuclease Inhibitor. The resulting amplimer was subcloned into the pGEM®-T Easy Vector and clones were purified with the Wizard® Plus SV Minipreps System. (2176)

Notes: Total RNA was isolated from human serum by combining 100µl of serum with 175µl of SV RNA Lysis Buffer. Only fresh or once-frozen serum was used. The SV Total RNA Isolation System was also used to isolate total RNA from tumor tissue. The rest of the protocol was followed as directed in the technical manual. The quantities of RNA isolated from serum were too low to quantitate so 1µl or 5µl of the isolated RNA was used in RT-PCR to analyze RNA content. (2160)

Notes: The SV Total RNA Isolation System was used to isolate total RNA from various rat tissues. The isolated RNA was used for RT-PCR. The authors also use a derivative of the pCI Vector called pCI-IRES-CD8 to express the TWIK protein specifically in COS cells. (2179)

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